Industrial production process for a vaccine against Japanese encephalitis virus and vaccine produced

ABSTRACT

A method is disclosed for industrially producing a Japanese encephalitis vaccine, wherein (a) cells from a cell line are cultured, (b) the resulting cell culture is inoculated with a Japanese encephalitis virus in the presence of a viral growth medium, (c) the virus is propagated and multiplied on the cells, (d) the viral growth medium is recovered in the form of a suspension of viruses produced by the cells, (e) the virus suspension is purified in at least one ion exchange chromatography step and a gel permeation step, and (f) the virus suspension is formulated and converted into a pharmaceutical form to preserve it until the moment of use. A Japanese encephalitis vaccine characterised in that it comprises a Japanese encephalitis virus produced by culturing cells from a cell line, and in that the cellular DNA content is less than 100 pg/dose, is also disclosed.

This is a National Stage of International Application No.PCT/FR96/01195, filed Jul. 29, 1996.

The present invention relates to a process for the production of avaccine for the prevention of Japanese encephalitis, based on Japaneseencephalitis virus (JEV) and in particular on a vaccine which can beused in man. The invention also relates to a vaccine obtained by thisprocess.

Japanese encephalitis virus, whose transmission vector is a mosquito, isthe cause of serious infections, known as Japanese encephalitides, inmany Far Eastern countries and in other areas of the world.

Vaccines against Japanese encephalitis are known, these being obtainedby processes which consist in injecting JEV intracranially into babymice and in harvesting the infected tissues. The tissue emulsionobtained is then purified, generally by precipitation methods, inparticular with protamine. Other techniques for purification of thesetissue preparations have also been proposed in the literature, such astechniques of ultrafiltration, of filtration and centrifugation, or ofprecipitation with polyethylene glycol, it being possible for thesetechniques to be combined with each other or with techniques of gelfiltration or of chromatography on cellulose sulphate or on crosslinkedpolysaccharide sulphate (JP-B-65,000,611, JP-A-53,133,627,JP-A-50,048,118, JP-A-2,223,531, U.S. Pat. No. 4,725,546,JP-A-49,020,322 and B-81,005,204, JP-B-67,025,408)

In the prior art, the viral preparations are inactivated, as recommendedby the World Health Organization, by chemical agents such asformaldehyde according to standardized procedures, namely long-terminactivation for 50 to 60 days at +4° C. at a formaldehyde concentrationof 1/2000, on account of the instability of the viruses at highertemperatures in this environment.

The commercial vaccines thus obtained are effective but are difficultand expensive to prepare, to purify and to inactivate. They may moreoverlead to side reactions due to contaminants originating from the babymouse tissues, thereby occasionally limiting their application.

It is thus desirable to be able to produce a vaccine by other moreindustrial techniques and in particular by using a multiplication and apropagation of the virus on a cell line. However, the production of avaccine in large quantities, using highly industrialized methods, ismuch more difficult than in the case of intracranial multiplication, notonly on account of the considerable quantities which must be dealt with,but also on account of the difficulties in obtaining and controllinghigh yields and also on account of the purification problems posed bycontaminants which are then encountered and which may originate from thecells or from the viral multiplication medium.

It is known in the prior art that Japanese encephalitis virus canpropagate on various cell cultures, including cultures of cell lines, inparticular Vero cells. However, the culturing methods disclosed do notmake it possible to obtain satisfactory yields under large-scaleindustrial culturing conditions, the only conditions allowing productionat moderate cost. Nor does the prior art describe any methods forpurifying to a high degree the viral preparations originating frompropagation and multiplication on cell lines.

The invention thus proposes to overcome these drawbacks and to provide aprocess for the production of a vaccine against Japanese encephalitis,which process may be used on a large scale and under safe, rapid andeconomic conditions and makes it possible to obtain an effective vaccineof very high purity in a very good industrial yield.

In order to achieve these aims, the subject of the invention is aprocess for the industrial production of a vaccine against Japaneseencephalitis, characterized in that it comprises the following steps:

a) culturing of cells originating from a cell line,

b) inoculation of the cell culture obtained by Japanese encephalitisvirus in the presence of a viral multiplication medium,

c) propagation and multiplication of the virus on the cells,

d) harvesting of the viral multiplication medium constituting asuspension of viruses produced by the cells,

e) purification of the viral suspension by at least one ion-exchangechromatographic step and a gel permeation step,

f) formulation and incorporation of the viral suspension in apharmaceutical form in order to ensure its conservation up to the timeof its use.

According to a particular feature of the invention, the amount of virusinoculated corresponds to a multiplicity of infection of less than 0.1.Thus, a good yield is obtained for the viral multiplication andpropagation.

According to a particular feature of the invention, the process alsocomprises a step of inactivation of the viral suspension before or afterthe purification step e). It is thus possible to manufacture aninactivated vaccine while at the same time using a virulent strain forthe viral multiplication and propagation.

According to a feature of the process of the invention, the inactivationis performed using a chemical agent at room temperature. Theinactivation thus takes place rapidly.

According to a particular embodiment, the process consists in using aVero cell line in order to ensure the viral multiplication. It is thuspossible to obtain good yields since these cells are very permissive toJapanese encephalitis virus.

According to another embodiment feature of the invention, the processconsists in purifying the viral suspension by carrying out the followingsteps:

ion-exchange chromatography,

adsorption chromatography,

gel permeation.

It is thus possible to obtain a vaccine with a very high degree ofpurity.

According to another embodiment of the invention, the process alsoconsists, after the harvesting step d), in reintroducing new viralmultiplication medium, in waiting for a sufficient period to allowfurther multiplication of the virus and in carrying out a furtherharvesting of the viral multiplication medium.

It is thus possible to obtain a considerable number of harvests from thesame cell culture.

The subject of the invention is also a vaccine obtained by culturing oncells originating from a cell line, characterized in that it includesJapanese encephalitis virus and in that the cellular DNA content is lessthan 100 pg/dose.

Such a vaccine has both the efficacy and the safety necessary, both fromthe point of view of the viral contaminants and of the proteins, to beadministered systematically to anybody liable to be in contact with thevirus.

Other subjects and advantages of the present invention will emerge onreading the description which follows.

According to the invention, the cell culture may be prepared either in afermenter or traditionally in flasks (Roux dishes, rolling flasks,Multitray™, Cell-Cube™, etc.).

Preferably, however, a large-volume fermenter (500 to 2000 l) containingmicrocarriers is used along with a cell culture medium, into which aninoculum of cells from a cell line permissive to Japanese encephalitisvirus is introduced; this cell line may in particular be BHK21 (BabyHamster Kidney) cells or alternatively Vero cells.

The microcarriers allowing the culturing in suspension in the fermentermay be various microcarriers already known for such a use; mention maybe made in particular of Cytodex 1™ particles at a concentration of 1 to3 g/l of culture medium as being particularly suitable for culturingVero cells. The durations, temperatures and other culturing conditions,and in particular the composition of the culturing medium, are adaptedas a function of the nature of the Vero cells on Cytodex 1™microcarriers, a duration of 4 days was suitable in order to obtain goodcell growth and thus make it possible to inoculate the virus before thestationary phase of the culture. The culture medium is then replaced bya viral multiplication medium and an inoculum of Japanese encephalitisvirus is introduced into the fermenter in an amount calculated to have alow value for the multiplicity of infection (or MOI), which is the ratioof the amount of viral particles introduced to the number of cellspresent.

This multiplicity is preferably less than about 0.1 and more preferablyless than 0.01.

The viral strain used may be an attenuated strain such as the strain SA14-14-2 or any known virulent immunogenic strain, such as the Nakayamaor Beijing strain. The strain P₃ at the 88th passage supplied by theNVSI (National Vaccine and Serum Institute, Beijing) is well suited tothe needs of the invention.

The medium used for the viral multiplication is a usual medium, such asMEM, in which it is important that the amount of proteins is reduced asmuch as possible. A medium whose concentration of proteins, usuallyhuman albumin, is less than 5 g/l is preferably used. Equallypreferably, a culture medium completely lacking proteins is used.

The period required for viral multiplication and propagation may bedetermined by monitoring the infectious titre. Harvesting of viruses isconsidered possible when the LD 50/ml titre is about 10⁷ or 10⁸. Theharvesting is carried out by simple removal of the viral multiplicationmedium which contains the viruses produced by the cells. Advantageously,after having removed the viral multiplication medium, new medium isreintroduced into the fermenter so as to allow a further viralmultiplication leading to a further harvest. It is thus readily possibleto obtain up to 8 successive harvests in the same fermenter from thesame cell culture. The period required for propagation andmultiplication of the virus in order to obtain an LD 50/ml assay of 10⁷or 10⁸ is generally from 2 to 3 days; a first harvest is preferablyperformed 3 days after the viral inoculation, followed by successiveharvests every 2 or 3 days.

Thus, a complete cycle in a fermenter may last for 23 days divided up asfollows:

D0: commencement of the culturing of cells on the microsupports in thefermenter,

D4: replacement of the cell culture medium by viral multiplicationmedium and inoculation of the virus,

D7: first harvest,

D9: second harvest,

D11: third harvest,

D14: fourth harvest,

D16: fifth harvest,

D18: sixth harvest,

D21: seventh harvest,

D23: eighth harvest.

The various harvests obtained may then be processed separately or as amixture.

The processing consists either of a purification alone or of apurification and an inactivation, it being possible for the inactivationto be performed before or after the purification step.

The inactivation is an essential step in the process according to theinvention when the viral strain used at the start is a virulent strain;on the other hand, when the strain used for the viral multiplication isan attenuated strain such as the strain SA 14-14-2, this inactivationstep may either be omitted or performed so as to be under the safestpossible conditions.

According to the invention, the inactivation is carried out usingchemical agents at room temperature. According to the invention, roomtemperature refers to a temperature considerably higher than +4° C.which is the temperature usually used by inactivation of JEV. Thistemperature may advantageously be between 20 and 37° C. and atemperature of about 25° C. is preferred. Indeed, it has been observedthat the virus is rapidly inactivated at this temperature and it hasmoreover been possible to note that, surprisingly, the virus containedin its viral multiplication medium is stable despite the hightemperature; this very short duration of the inactivation is animportant advantage of the process of the invention from an industrialpoint of view.

The chemical agents used for the inactivation may in particular beformaldehyde or β-propiolactone; formaldehyde is preferred. Theinactivation may be carried out, for example, using formaldehyde at 25or at 37° C. for 14 days or less; the duration will preferably be atleast 7 days. Advantageously, according to the invention, theformaldehyde concentration used may be less than that usedconventionally in the inactivation of JEV; it may, for example, be fromabout 1/2000 to 1/8000 and in particular 1/4000.

It is also possible to carry out 2 successive and different inactivationsteps using, for example, 2 different chemical agents.

It is also possible, before this inactivation step, to filter eachharvest so as to remove cell debris (proteins, nucleic acids, etc.), aswell as to concentrate each one so as to increase the viral titre andthe protein content of the liquid medium. The concentration factor ispreferably at least equal to 10, for example about 1000. Concentratingmay be carried out by the usual means and in particular byultrafiltration.

The viral suspension, which may or may not be inactivated depending onthe process used, must be purified. According to an important feature ofthe invention, the purification step comprises at least onechromatography step. Advantageously, the following 3 steps are carriedout successively:

ion-exchange chromatography

adsorption chromatography

gel permeation.

The ion-exchange chromatography is preferably a chromatographic exchangeof anions, whether weak or strong ones. The support used is, forexample, DEAE-Spherodex™ (sold by Biosepra, USA) which selectivelyretains the viral particles and allows the bulk of the contaminantproteins to pass through.

The eluate containing the viruses may then be carried out on supportssuch as hydroxyapatite or chelating gels (calcium-chelating, etc.). Inthis case, the virus does not become bound to the support, which moreespecially retains the nucleic acids.

After this step, gel filtration or molecular sieving (again called gelpermeation) is carried out on any suitable support such as Sepharose6FF™ (Pharmacia) or Fractogel™ (E.Merck). During this operation, elutionmakes it possible to recover the viral particles in the first fraction;the following elution peaks correspond to the viral proteins and to theresidual impurities. Thus, for the manufacture of a vaccine, the firstfraction only of the eluate is conserved.

Advantageously, between the adsorption chromatography and the gelfiltration, a step of concentrating may be carried out, preferably byultrafiltration with a membrane whose cut-off threshold is 10,000daltons.

The purified and optionally inactivated viral suspension is thenformulated in order to obtain the desired antigenic titre; it is alsopossible to add a stabilizer or an adjuvant thereto; it is subsequentlymade into a pharmaceutical form so as to be conserved under goodconditions until the time of its use.

EXAMPLE 1. Materials

Vero Cells: the Vero cells used to inoculate the fermenter are derivedfrom a Vero cell bank at the 137th passage, this bank having undergoneall the checks necessary for its characterization and qualification.

The JEV: the viral strain used is the strain P₃ at the 88th passagesupplied by the NVSI.

A received vial of virus is suspended in 100 ml of medium and filteredusing a 0.1 μm filter. The solution is used to infect two 75 cm² flasks.Five days later, the supernatant collected is filtered (0.2 μm filter).Several harvests were made and the mixture forming the primary seedingbatch has an LD 50/ml titre =10⁸.16. The working seeding batch isprepared in a proportion of 10 ml of primary seeding batch to infect 12850 cm² rolling flasks. Several harvests may be taken and the mixture of30.2 liters has an LD 50/ml titre=10⁸.31.

The primary and working seeding batches are subsequently checked so asto ensure their characterization and qualification.

2. Culturing process

The 500 l fermenter tank is filled with a conventional culture mediumfor Vero cells containing Cytodex 1™ microcarriers at a concentration of3 g/l. The medium is seeded with an inoculum of Vero cells (200,000cells/ml).

The cells are left to attach and to grow for four days. At the end ofthe four days, the culture medium is replaced by a viral multiplicationmedium.

A viral inoculum is introduced into the tank with an amount of viruscalculated so as to have a multiplicity of infection MOI equal to 1/500.The various cultures are prepared at a temperature of 37° C. Three daysafter the viral inoculation, the viral multiplication medium iscollected, this giving the first harvest. The viral multiplicationmedium is replaced by a new medium and further harvesting is carried outevery other day, the total number of harvests being equal to six.

Table I shows the titres obtained at each harvest.

                  TABLE I                                                         ______________________________________                                        Harvest No.                                                                            H1      H2      H3    H4    H5    H6                                 ______________________________________                                        Days after                                                                             3       5       7     10    12    14                                 infection                                                                     Titre    10.sup.8.4                                                                            10.sup.8.4                                                                            10.sup.8.5                                                                          10.sup.8.1                                                                          10.sup.8.2                                                                          10.sup.7.3                         LD 50/ml                                                                      ______________________________________                                    

The harvests are filtered on a membrane filter (pore diameter: 0.2 μm).

The harvests are all mixed together.

The harvest mixture is concentrated by a factor of 100 byultrafiltration on a 10,000 dalton membrane.

A formaldehyde solution at a final concentration of 1/4000 is added tothe concentrated harvest mixture and the resulting mixture is maintainedat room temperature (from 20 to 25° C.) with continuous stirring for 14days.

A further filtration is then carried out and the inactivation isverified by a two-step check according to the WHO procedure (TechnicalReport 771 from 1988).

All the preparations inactivated with formaldehyde proved to besatisfactory.

The inactivated solution is passed through an ion-exchangechromatography column containing DEAE groups equilibrated at pH 8(DEAE-Spherodex™ resin). The virus is retained on the column. Afterwashing with a phosphate buffer, the virus is eluted with a phosphatebuffer, 0.2 M NaCl. Most of the protein contaminants are thus removed.

The eluate is purified by chelation affinity by calcium interaction, byinjecting the eluate through a Chelating Sepharose™ column (Pharmacia).The virus is not bound and travels directly through the column.

Concentrating is then carried out by ultrafiltration on membranes whosecut-off threshold is 10,000 Da, in order to reduce the volume of theviral suspension by a factor of about 20.

The concentrated solution of pre-purified virus is introduced onto aSepharose 6FF™ column. An elution is carried out with a 0.2 M NaClphosphate buffer. The viral particles are eluted in the excludedfraction.

The characteristics of the purification process are featured in TableII.

                  TABLE II                                                        ______________________________________                                        Step         DEAE          Chelation                                                                            Gel filtration                              ______________________________________                                        Residual     50%           100%   0.4%                                        proteins (*)                                                                  DNA content  <7000         <30    <30                                         (pg/ml)                                                                       Virus recovered                                                                            90%            76%    20%                                        (*) (ELISA                                                                    protein E)                                                                    ______________________________________                                         (*) Stepwise yields                                                      

The purified inactivated viral solution was used to immunize mice byinjection on day 0 and then on day 7. The test with the virulent virusis carried out using a solution containing 10⁵ LD 50/ml on day 30. Allthe mice vaccinated with the purified undiluted preparation and with thepurified preparation diluted to 1/32 were protected. In the same test,the mice vaccinated with Biken vaccine (1/32 dilution) were tested andonly 2/5 of them were protected.

What is claimed is:
 1. A process for the industrial production of avaccine against Japanese encephalitis, comprising:a) culturing cellsfrom a cell line on a cell culture medium, b) inoculating the cells withJapanese encephalitis virus in the presence of a viral multiplicationmedium, c) propagating and multiplying the virus, d) harvesting theviral multiplication medium, e) purifying viruses from the viralmultiplication medium by at least one ion-exchange chromatographic step,followed by adsorption chromatography and gel permeation, and f)formulating and incorporating the viruses from the viral multiplicationmedium in a pharmaceutical form.
 2. The process according to claim 1,wherein the amount of virus inoculated corresponds to a multiplicity ofinfection (MOI) of less than 0.1.
 3. The process according to claim 1,further comprising inactivating the viruses harvested from the viralmultiplication medium before or after purification.
 4. The processaccording to claim 3, wherein the inactivation is performed using achemical agent at room temperature.
 5. The process according to claim 1,wherein the cells are Vero cells.
 6. The process according to claim 1,further comprising reintroducing new viral multiplication medium afterthe harvesting and repeating c)-f).
 7. The process according to claim 6,wherein the reintroducing of the new viral multiplication medium andsubsequent repeating of c)-f) is performed 2, 3, 4, 5, or 6 times. 8.The process according to claim 1, further comprising filtering the viralmultiplication medium after the harvesting and before the purifying. 9.The process according to claim 1, wherein the viral multiplicationmedium possesses a protein concentration of less than 5 g/l.
 10. Avaccine against Japanese encephalitis obtained by the method of claim 1,wherein the cellular DNA content of the vaccine is less than 100pg/dose.